Botulinum toxins (BoNTs) are among the most potent toxins known, thus defining it as a Class A bio-terrorism threat by NIAID. Intoxication with BoNT leads to flaccid paralysis, respiratory arrest, and death by blocking acetylcholine release at neuromuscular junctions. BoNTs mediate paralysis by cleaving molecules found in synaptic terminals, synaptosomal-associated protein of 25 kD (SNAP-25), vesicle-associated membrane protein (VAMP), or syntaxin. Early treatment with humanized antibodies can prevent progression and death by BoNT intoxication. However, many surviving patients exhibit a persistent or chronic fatigue syndrome. Unfortunately, not much is known about the cellular and molecular mechanism of this fatigue syndrome. A prime hypothesis here is that this disabling fatigue syndrome is caused by cell injury and death of motoneurons and that different BoNT serotypes have differential effects in mediating this newly recognized neurodegenerative disorder. Thus the goal of this project will be to gain new insight into how BoNTs mediate motoneuron cell death. To achieve this goal, primary motoneurons will be analyzed using approaches such as cell biology, immunocytochemistry, time-lapse deconvolution microscopy, electron microscopy, and gene and peptide transfer. Among the specific questions that will be addressed are: (1) How does BoNT/C but not BoNT/A induce damage to synaptic endings and neurites, eventually resulting in motoneuron degeneration? (2) What are the real-time changes in synaptic endings and neurites, cytoskeleton, and mitochondria linked to BoNT-induced motoneuron cell death? (3) Does cleavage of SNAP-25, syntaxin, or synaptobrevin/VAMP suffice to induce motoneuron damage and cell death initiated by BoNTs? Results obtained here will in general lead to a better understanding of the mode of action of BoNTs. Most importantly, new insights gained here will set the foundation to treat and prevent long-term, devastating neurological effects linked to BoNT-mediated intoxication after a bioterrorism attack.